Microstimulation has been an invaluable tool for neuroscience researchers to infer functional connections between brain structures or causal links between structure and behavior. In recent years, therapeutic microstimulation is gaining interest for the restoration of visual, auditory and somatosensory functions as well as emerging applications in bioelectronic medicine. Current neural stimulation parameters and safety limits need to be revised for microelectrodes using more systematic and advanced methodologies. Stimulations via microelectrodes often require high charge injection for effective modulation of neural tissue without exceeding the threshold to harm the tissue or the electrodes. Therefore, advanced electrode materials with high charge injection capability and stability are highly desired. We have developed several types of stimulation materials based on conducting polymer PEDOT and nanomaterial composites. These materials present different charge transfer and electrochemical properties as well as biocompatibility, and the effects of these properties on microstimulation have yet to be comprehensively characterized. This proposal aims to establish new in vitro and in vivo models to examine the efficiency and safety of stimulation via multiple electrode materials, ranging from the clinically approved Pt and Iridium Oxide (IrOx) to the emerging PEDOT nanocomposites. Another challenge with micro-stimulation is its sensitivity to host tissue responses. Implantation of electrodes causes electrode fouling, progressive neuronal loss and inflammatory gliosis immediately surrounding the implants. Loss of nearby neurons and axons leads to decreased stimulation efficacy, while electrode fouling and gliosis increase impedance. Additionally, stimulation itself may further exacerbate host tissue responses if above the safety limit, which has yet to be defined for microelectrodes and emerging electrode materials. Using in vivo imaging in fluorescently labeled mice, we will examine the acute and chronic effects of microstimulation on neurons, microglia and vasculature, while monitoring the electrode material and electrochemical products. We will use an in vitro multielectrode arrays (MEA) system to study the effects of electrical stimulation on material and cells, in order to pinpoint the mechanisms of material and tissue damage. The first aime is to assess the efficiency and safety limit of neural stimulation via different electrode materials in vivo in acute experiments. For efficiency testing, we will implant the electrodes in the cortices of GCaMP mice and use 2-photon microscopy to image the calcium signal in order to determine stimulation threshold and optimum stimulation parameter for each electrode material. as a function of stimulation parameters. Stimulation threshold and efficiency for different pulse width, interphase period, bias potential and frequency from each electrode material type will be determined. For safety testing, we will use Syn-RCaMP/Cx3Cr1-GFP mice to visualize both neuronal and microglia cells and determine the damage threshold. The second aim is to examine the effects of stimulation on electrode materials and cultured cells in vitro. Using a high-throughput in vitro MEA system in which the six microelectrode materials can be deposited, we will stimulate at safe and unsafe parameters (identified in vivo from Aim 1) for up to 12 weeks. We will assess electrode material stability and analyze the stimulated media to identify electrochemical and degradation products. The toxicity of stimulated media will be tested in cultures of neuron, microglia, endothelial cells and neuron-microglia co-culture at varying doses to determine the detrimental effects of electrochemical and degradation products on these cells. Finally, we will directly stimulate the cells cultured on MEAs and characterize cell behavior using quantitative RNA and protein analysis, neural recording/stimulation and immunohistochemistry. The third aim is to characterize the chronic safety and stability of microstimulation in vivo from different electrode materials. Stimulation will be applied one hour per day to microelectrode arrays chronically implanted in Syn-RCaMP/Cx3Cr1-GFP animals for 12 weeks. In each weekly imaging session, we will measure the in vivo impedance, CV, charge injection limit, and stimulation threshold. The neuronal response (activity, health, density), microglia (morphology, coverage and motility) and BBB integrity will be recorded, and compared over time points between material types, and to the non-stimulated sites. In addition, we will closely track the electrode health with electrochemical interrogation, imaging and explant analysis.